Prion diseases or transmissible spongiform encephalopathies are infectious neurodegenerative diseases of humans and animals. A major feature of prion diseases is the refolding and aggregation of a normal host protein, prion protein (PrP), into a disease-associated protease-resistant form (PrPres) which may contribute to brain damage. There is a strong species barrier which prevents or delays cross-species prion infection, and this barrier mostly depends on amino acid sequence differences in the PrP of various species. For example, humans appear to be resistant to sheep scrapie in spite of extensive epidemiological evidence of exposure of humans to scrapie-infected sheep tissues. Similarly mouse-adapted sheep scrapie strains are not known to infect transgenic mice expressing human PrP, whereas these mice are susceptible to prion strains from humans with sporadic Creutzfeldt-Jakob disease. In FY15 we tested the effect of the presence of the prion protein C-terminal glycophosphatidylinositol (GPI) membrane anchoring group on the prion species barrier between mice and humans. Prion stocks of mouse-adapted sheep scrapie (strain 22L) were generated by infection of mice expressing PrP with or without the anchoring GPI moiety. Similar mouse infectious titers of these stocks were then tested by intracerebral injection of various dilutions in tg66 transgenic mice expressing human PrP, and mice were followed for signs of clinical scrapie. No disease was observed in tg66 mice given prions from mice expressing normal anchored PrP. However, tg66 mice were susceptible to prions from mice expressing anchorless PrP. Thus the lack of the GPI anchor group on PrP appeared to reduce the effect of the mouse-human species barrier. This result was dependent on high expression of human PrP in tg66 mice and was not seen in mice expressing human PrP at lower levels. In FY15 we also studied the neuroinflammatory response in mouse brain at preclinical time-points after infection with 2 different strains of mouse prions (22L and RML). Alterations in gene regulation were quantitated by analysis of brain RNA using quantitative real-time RT-PCR with SuperArray kits focusing on genes involved in inflammatory responses, such as cytokines, cytokine receptors, JAK/STAT and IL6/STAT3 pathways. These experiments detected upregulation of 15 previously unreported genes in these arrays, and many of which were also confirmed by quantitative protein analysis. The results indicated that a prominent neuroinflammatory response occurred starting at 70 days post-infection for scrapie strains 22L and RML. Similar genes were upregulated with both strains even though these strains are known to infect different brain regions. The finding that this response was detectable prior to clinical signs and vacuolar pathology suggested that neuroinflammation might contribute to the pathogenic damage process, and might be a possible target for therapeutic intervention.